Assay methods for state-dependent calcium channel agonists/antagonists

ABSTRACT

Methods of identifying activators and inhibitors of voltage-gated ion channels are provided in which the methods employ cells transfected with a voltage-gated ion channel of interest and a corollary channel to control the membrane potential of the cells by changing extracellular ion concentration. This allows for more convenient, more precise experimental manipulation of these transitions, and, coupled with efficient methods of detecting the result of ion flux through the channels, provides methods that are especially suitable for high throughput screening.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/418,017, filed Oct. 10, 2003, the contents of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention is directed to methods and cells for studying theeffect of candidate compounds on the activity of calcium channels. Themethods utilize cells that express a calcium channel of interest andwhich express a potassium channel. The engineered cells allow for finecontrol of the membrane potential of the cells, which, in turn, providea high resolution assay for studying the effects of targeted compoundsat various states of the calcium channel.

BACKGROUND OF THE INVENTION

Certain molecular events in eukaryotic cells depend on the existence ormagnitude of an electric potential gradient across the plasma (i.e.,outer) membrane of the cells. Among the more important of such events isthe movement of ions across the plasma membrane through voltage-gatedion channels. Voltage-gated ion channels form transmembrane pores thatopen in response to changes in cell membrane potential and allow ions topass through the membrane. Voltage-gated ion channels have manyphysiological roles. They have been shown to be involved in maintainingcell membrane potentials and controlling the repolarization of actionpotentials in many types of cells (Bennett et al., 1993, CardiovascularDrugs & Therapy 7:195-202; Johnson et al., 1999, J. Gen. Physiol.113:565-580; Bennett & Shin, “Biophysics of voltage-gated sodiumchannels,” in Cardiac Electrophysiology: From Cell to Bedside, 3^(rd)edition, D. Zipes & J. Jalife, eds., 2000, W.B. Saunders Co., pp. 67-86;Bennett & Johnson, “Molecular physiology of cardiac ion channels,”Chapter 2 in Basic Cardiac Electrophysiology and Pharmacology, 1^(st)edition, A. Zasa & M. Rosen, eds., 2000, Harwood Academic Press, pp.29-57). Moreover, mutations in sodium, calcium, or potassiumvoltage-gated ion channel genes leading to defective channel proteinshave been implicated in a variety of disorders including the congenitallong QT syndromes, ataxia, migraine, muscle paralysis, deafness,seizures, and cardiac conduction diseases, to name a few (Bennett etal., 1995, Nature 376:683-685; Roden et al., 1995, J. Cardiovasc.Electrophysiol. 6:1023-1031; Kors et al., 1999, Curr. Opin. Neurol.12:249-254; Lehmann et al., 1999, Physiol. Rev. 79:1317-1372; Holbauer &Heufelder, 1997, Eur. J. Endocrinol. 136:588-589; Naccarelli &Antzelevitch, 2000, Am. J. Med. 110:573-581).

Several types of voltage-gated ion channels exist. Voltage-gatedpotassium channels establish the resting membrane potential and modulatethe frequency and duration of action potentials in neurons, musclecells, and secretory cells. Following depolarization of the membranepotential, voltage-gated potassium channels open, allowing potassiumefflux and thus membrane repolarization. This behavior has madevoltage-gated potassium channels important targets for drug discovery inconnection with a variety of diseases. Dysfunctional voltage-gatedpotassium channels have been implicated in a number of diseases anddisorders. Wang et al., 1998, Science 282:1890-1893 have shown that thevoltage-gated potassium channels KCNQ2 and KCNQ3 form a heteromericpotassium ion channel known as the “M-channel.” Mutations in KCNQ2 andKCNQ3 in the M-channel are responsible for causing epilepsy (Biervert etal., 1998, Science 279:403-406; Singh et al., 1998, Nature Genet.18:25-29; Schroeder et al., Nature 1998, 396:687-690).

Voltage-gated sodium channels are transmembrane proteins that areessential for the generation of action potentials in excitable cells(Catterall, 1993, Trends Neurosci. 16:500-506). In mammals,voltage-gated sodium channels consist of a macromolecular assembly of αand β subunits with the α subunit being the pore-forming component αsubunits are encoded by a large family of related genes, with some αsubunits being present in the central nervous system (Noda et al., 1986,Nature 322:826-828; Auld et al., 1988, Neuron 1:449-461; Kayano et al.,1988, FEBS Lett. 228:187-194) and others in muscle (Rogart et al., 1989,Proc. Natl. Acad. Sci. USA 86:8170-8174; Trimmer et al., 1989, Neuron3:3349).

Voltage-gated calcium channels are transmembrane proteins that in theopen configuration allow the passive flux of Ca²⁺ ions across the plasmamembrane, down the electrochemical gradient. They mediate various cellfunctions, including excitation-contraction coupling, signaltransduction, and neurotransmitter release. Three major classes ofcalcium channel antagonists including the dihydropyridines,benzothiazepines and phenylalkylamines have been widely used clinicallyin the treatment of cardiovascular diseases. These drugs antagonize theL-type calcium channels found throughout the body, including thecardiovascular system. Calcium channels are allosteric proteins thatundergo changes in conformational state. The distinct conformationalstates of these proteins have different affinities for ligands,including these antagonists. Membrane potential is an allostericeffector of these conformational changes in ion channel proteins. Thepotency of inhibition by these calcium channel antagonists is dependenton the state of the calcium channel. Previously studies onstate-dependent interactions of these antagonists were identifiedthrough voltage clamp (1), radioligand binding (2) and cell based, e.g.smooth muscle contraction (3) studies. While each of these methodsyields valuable information each has its drawbacks in terms ofinformation content or throughput, respectively.

Calcium channels are membrane-spanning, multi-subunit proteins thatallow controlled entry of Ca+2 ions into cells from the extracellularfluid. Cells throughout the animal kingdom, and at least some bacterial,fungal and plant cells, possess one or more types of calcium channel.

The most common type of calcium channel is voltage dependent. Most“excitable” cells in animals, such as neurons of the central nervoussystem (CNS), peripheral nerve cells and muscle cells, including thoseof skeletal muscles, cardiac muscles, and venous and arterial smoothmuscles, have voltage-dependent calcium channels. “Opening” of avoltage-dependent channel to allow an influx of Ca+2 ions into the cellsrequires a depolarization to a certain level of the potential differencebetween the inside of the cell bearing the channel and the extracellularenvironment bathing the cell. The rate of influx of Ca+2 into the celldepends on this potential difference.

Multiple types of calcium channels have been identified in mammaliancells from various tissues, including skeletal muscle, cardiac muscle,lung, smooth muscle and brain, [see, e.g., Bean, B. P. (1989) Ann. Rev.Physiol. 51:367-384 and Hess, P. (1990) Ann. Rev. Neurosci. 56:337]. Thedifferent types of calcium channels have been broadly categorized intofive classes, L-, T-, N-, P/Q and R-type, distinguished by currentkinetics, holding potential sensitivity and sensitivity to calciumchannel agonists and antagonists.

Current methods of drug discovery often involve assessing the biologicalactivity (i.e., screening) of tens or hundreds of thousands of compoundsin order to identify a small number of those compounds having a desiredactivity. In many high throughput screening programs, it is desirable totest as many as 50,000 to 100,000 compounds per day. Unfortunately,current methods of assaying the activity of voltage-gated ion channelsare ill suited to the needs of a high throughput screening program.Current methods often rely on electrophysiological techniques. Standardelectrophysiological techniques involve “patching” or sealing againstthe cell membrane with a glass pipette followed by suction on the glasspipette, leading to rupture of the membrane patch (Hamill et al., 1981,Pflugers Arch. 391:85-100). This has limitations and disadvantages.Accessing the cell interior may alter the cell's response properties.The high precision optical apparatuses necessary for micromanipulatingthe cells and the pipettes make simultaneous recording from more than afew cells at a time impossible. Given these difficulties, the throughputthat can be achieved with electrophysiological techniques falls farshort of that necessary for high throughput screening.

Various techniques have been developed as alternatives to standardmethods of electrophysiology. For example, radioactive flux assays havebeen used in which cells are exposed with a radioactive tracer (e.g.,⁸⁶Rb⁺, ²²Na⁺, [¹⁴C]-guanidinium and ⁴⁵Ca) and the flux of theradio-labled ion is monitored. Cells loaded with the tracer are exposedto compounds and those compounds that either enhance or diminish theefflux of the tracer are identified as possible activators or inhibitorsof ion channels in the cells' membranes.

Assays that measure the change in a cell's membrane potential due to thechange in activity of an ion channel have been developed. Such assaysoften employ voltage sensitive dyes that redistribute between theextracellular environment and the cell's interior based upon a change inmembrane potential and that have a different fluorescence spectrumdepending on whether they are inside or outside the cell. A relatedassay method uses a pair of fluorescent dyes capable of fluorescenceresonance energy transfer to sense changes in membrane potential. For adescription of this technique, see González & Tsien, 1997, Chemistry &Biology 4:269-277. See also Gonzalez & Tsien, 1995, Biophys. J.69:1272-1280 and U.S. Pat. No. 5,661,035. Other methods employ ionselective indicators such as calcium dependent fluorescent dyes tomonitor changes in Ca²⁺ influx during opening and closing of calciumchannels.

Ideally, methods of screening against voltage-gated ion channels requirethat the transmembrane potential of the cells being assayed becontrolled and/or that the ion channels studied be cycled between openand closed states. This has been done in various ways. In standardelectrophysiological techniques, the experimental set-up allows fordirect manipulation of membrane potential by the voltage clamp method(Hodgkin & Huxley, 1952, J. Physiol. (Lond.) 153:449-544), e.g.,changing the applied voltage. In other methods, changing theextracellular K⁺ concentration from a low value (e.g., 5 mM) to a highervalue (e.g., 70-80 mM) results in a change in the electrochemicalpotential for K⁺ due to the change in the relative proportion ofintracellular and extracellular potassium. This results in a change inthe transmembrane electrical potential towards a more depolarized state.This depolarization can activate many voltage-gated ion channels, e.g.,voltage-gated calcium, sodium, or potassium channels. Alternatively, Na⁺channels can be induced into an open conformation by the use of toxinssuch as veratridine or scorpion venom (Strichartz et al., 1987, Ann.Rev. Neurosci. 10:237-267; Narahashi & Harman, 1992, Meth. Enzymol.207:620-643). While sometimes effective, such experimental manipulationsmay alter the channel pharmacology, can be awkward to perform, and canlead to artifactual disturbances in the system being studied.

Electrical field stimulation (EFS) has been used to activate ionchannels. In this approach, membrane potential is altered but notcontrolled. The uncertainty and lack of control of membrane potentialmake EFS a less than optimal method for the study of ion channels.

HEK293 cells have been grown on a silicon chip made up of an array offield-effect transistors. Some of the cells were positioned over thegate region of the transistors, thus having portions of their plasmamembranes overlying the source and the drain. When a patch pipette insuch cells manipulated the intracellular voltage, Maxi-K potassiumchannels in the cells' plasma membranes were opened. This led to currentflow in the region between the cells' membrane and the transistor. Thiscurrent flow modulated the source-drain current, which could be detectedby an appropriate device. The chip plus cells was said to have potentialas a sensor and as a prototype for neuroprosthetic devices. See Straubet al., 2001, Nature Biotechnol. 19:121-124; Neher, 2001, NatureBiotechnol. 19:114.

SUMMARY OF THE INVENTION

The present invention is directed to methods of identifying activatorsand inhibitors of voltage-gated ion channels, and specifically calciumion channels. The methods employ cells transformed to express avoltage-gated calcium ion channel of interest and an inward rectifierpotassium channel. The addition of the potassium channel allows for thefine control of the membrane potential of the cells. Manipulation of theextracellular potassium concentration controls the membrane potentialwhich in turn affects the open/close state transitions of thevoltage-gated ion channels. This allows for more convenient, moreprecise manipulation of these transitions, and, coupled with efficientmethods of detecting ion flux, results in methods that are especiallysuitable for high throughput screening in order to identify substancesthat are channel state dependent modulators of voltage-gated ionchannels.

According to a specific embodiment, the present invention describes thestate-dependent interactions of the calcium channel antagonists directlyin a functional cell-based FLIPR (Fluorometric Imaging Plate Reader)assay, which measures calcium influx through a voltage-dependent calciumchannel (VDCC). The cell line used in this embodiment has a stablytransfected L-type calcium channel, the α1C subunit. It also wastransfected with the Kir 2.3 inward rectifier K channel, which allowsfor control of cell membrane potential through alteration ofextracellular [K⁺]o. Preincubation of the cells for 10 min in 30 mM[K⁺]o partially depolarizes the cells. The inhibitory effect of calciumchannel antagonists on calcium influx in response to a high [K⁺]odepolarization (final [K⁺]o 85.8 mM) was shifted to the left comparedwith that observed for cells in normal, physiological [K⁺]o (5.8 mM).The ratio of IC₅₀ values between the potencies for the antagoniststested in the normally polarized and depolarized cells was 4 to 20-fold.The results suggest that the interaction of these calcium channelantagonists with the channel expressing cells is dependent upon thestate of the channel, which is modulated by changes in membranepotential. The state dependent assay demonstrated in these studies isuseful for evaluating state dependent inhibitory potency of a largenumber of samples and can be used to identify state-dependent calciumchannel antagonists.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows immunostaining of alpha IC subunit in the wild type HEK 293cells and cells stably transfected with the L-type α1C channel(C1-6-37-3).

FIG. 2 shows immunostaining of Kir2.3 subunit in the wild type HEK293cells and the cells stably transfected with the L-type α1C channel(C1-6-37-3).

FIG. 3 is a graph showing the relationship between extracellularpotassium ([K]o) and cell membrane potential. Three situations areshown. One is the prediction of the Nernst equation for a perfectlyK-selective membrane. The other curves show the effects of partialpermeability by other ions, Na⁺ and/or Cl⁻. Membrane potential can beset in a non-voltage clamped cell by adjusting external potassium.

A cell line expressing an inward rectifier K channel (Kir2.3) to set theresting membrane potential will permit control of membrane restingpotential by extracellular potassium.

FIG. 4 is a graph demonstrating the dose-response relationship forK⁺-stimulated calcium influx in wild type HEK 293 cells and cells stablytransfected with the L-type α1C channel (C₁₋₆-37-3).

FIG. 5 is a graph demonstrating a comparison of nimodipine andmibefradil inhibition curves in K⁺-stimulated calcium influx inC₁₋₆-37-3 cells under resting condition (5.8 mM K=−65 mV).

FIG. 6 is a graph representing the nimodipine inhibition curvestimulated by K⁺ (final 85.8 mM) either in 30 mM K⁺ (depolarizedcondition, −28 mV) or 5.8 mM K⁺ (resting condition, −65 mV).

FIG. 7 is summary table of IC50 (nM) values for calcium channelantagonists in 30 mM K⁺ (depolarized condition, −28 mV) and 5.8 mM K⁺(resting condition, −65 mV).

DETAILED DESCRIPTION OF THE INVENTION

Without intending to bound by any theory, voltage gated calcium channelsopen as a function of membrane potential such that the probability ofopening increases with membrane depolarization. Voltage gated calciumchannels inactivate (close/desensitize) as a function of membranepotential such that the probability of inactivation increases withmembrane depolarization. These steady state voltage dependent processesoverlap. Changes in membrane potential populate different conformationalstates of these channels (closed, open or inactivated). Drug binding tovoltage gated calcium channels is often channel state dependent suchthat more or less binding occurs depending upon the state occupied.Control of membrane potential, permits channels to be manipulated intovarious states. This membrane potential control is typically achieved byvoltage clamp electrophysiology methods, but this method is not atpresent amenable to high throughput drug screening.

Specifically exemplified herein is an assay to determine state-dependentdrug-calcium channel interactions using a cell line that co-expresses apotassium channel (Kir2.3) that determines the resting membranepotential of the cells as a function of the external potassium ionconcentration ([K]o) and a voltage gated calcium channel. Co-expressedin these cells is the L-type voltage gated calcium channel complex(alpha1C, alpha2-delta, beta2a). Potassium is used in a two step mannerin this assay. First it is used to set the resting membrane potential(Vm) during antagonist incubation. Two conditions were selected forillustration purposes, polarized and depolarized resting conditions. Inthe polarized resting condition, cells are incubated in 5.8 mM [K]o toset the membrane potential to −65 mV (Vm as a function of [K]o). Drugsexposed to these cells will bind to calcium channels primarily in theclosed, rested, low affinity state. In order to reveal higher affinitystates of the calcium channels, the cells are incubated in 30 mM [K]o,in order to chronically and partially depolarize them to −28 mV duringdrug exposure. This change in the membrane potential, shifts the calciumchannels into the higher affinity inactivated states and antagonistbinding is enhanced. Upon establishing these two different conditionsfor drug exposure, channels are then forced to open by furtherdepolarization to near 0 mV by exposure to 85.8 mM [K]o. Opening ofthese channels normally under control, non-antagonist exposedconditions, allows calcium influx into the cells. This calcium influx isdetected using a calcium sensitive dye (eg Fluo-3, Fluo4, Fura2, etc.).If the calcium influx is diminished by exposure to antagonists, thiswill be detected when compared to the control condition. In some cases,antagonists will bind with greater affinity to the channels in thedepolarized (30 mM [K]o) condition. In these cases, the same drug willappear more potent under these depolarized assay conditions. Thisapproach creates a novel high throughput calcium channels assay systemthat is capable of detecting and measuring calcium channel statedependent drug interactions as have been described using low throughputvoltage clamp measures on single cells.

This foregoing approach and the referenced cells have been tested usingconventional voltage- and current-clamp methods, and the membranepotential changes as a function [K]o and the state dependent calciumcurrent and drug affinities have been confirmed experimentally. Theforegoing approach can be modified as taught herein to studystate-dependencies of agonists/antagonists for many different types ofion channels.

In one embodiment, the present invention involves providing a substrateupon which living eukaryotic cells, preferably mammalian cells, arepresent where the cells express voltage-gated calcium ion channels intheir plasma membranes. Upon application of varying concentrations ofextracellular calcium, voltage-gated ion channels either open or close,thereby modulating the flow of at least one type of ion through theplasma membranes of the cells. This modulation of ion flow, or a changein membrane potential that results from the modulation of ion flow, isdetected, either directly or indirectly, preferably by the use offluorescent indicator compounds in the cells. Collections of substances,e.g., combinatorial libraries of small organic molecules, naturalproducts, phage display peptide libraries, etc., are brought intocontact with the voltage-gated ion channels in the plasma membranes ofthe cells and those substances that are able to affect the modulation ofion flow are identified. In this way, the present invention providesmethods of screening for activators and inhibitors of voltage-gated ionchannels, particularly calcium channels. Such activators and inhibitorsare expected to be useful as pharmaceuticals or as lead compounds fromwhich pharmaceuticals can be developed by the usual processes of drugdevelopment, e.g., medicinal chemistry.

Accordingly, the present invention provides a method for identifyingmodulators of the activity of a voltage-gated calcium ion channelcomprising:

-   -   (a) providing cells expressing the voltage-gated calcium ion        channel and expressing an inward rectifying potassium channel;    -   (b) dividing the cells into group 1 and group 2;    -   (c) changing extracellular potassium concentration of the group        2;    -   (c) exposing the cells of groups 1 and 2 to a substance of        interest;    -   (d) depolarizing the cells of groups 1 and 2 while monitoring        ion flux through the voltage-gated calcium ion channel;    -   (c) comparing the ion flow through the voltage-gated calcium ion        channel in groups 1 and 2;    -   where a difference in the ion flow through the voltage-gated        calcium ion channel in groups 1 and 2 indicates that the        substance is a modulator of the voltage-gated channels, and        where the potency of the modulator is affected by the state of        the voltage-gated calcium ion channel.

For the sake of simplicity, the above methods are described in terms of“a” voltage-gated ion channel although those skilled in the art willunderstand that in actual practice the cells will express a plurality ofthe voltage-gated ion channels for which modulators are sought.Generally, each cell will express at least 10², 10³, 10⁴, 10⁵, 10⁶ ormore molecules of the voltage-gated ion channel. Also, ion flow will bemonitored through the plurality of the voltage-gated ion channels ratherthan through a single voltage-gated ion channel. Similarly, the methodswill generally be practiced by employing a plurality of cells, eventhough the methods are described above in terms of “a” cell.

Generally, the methods of the present invention will be carried out on asubstrate that is a modified version of a standard multiwell tissueculture plate or microtiter plate.

The skilled person will recognize that it is generally beneficial to runcontrols together with the methods described herein. For example, itwill usually be helpful to have a control in which the substances aretested in the methods against cells that preferably are essentiallyidentical to the cells that are used in the methods except that thesecells would not express the voltage-gated ion channels of interest. Inthis way it can be determined that substances which are identified bythe methods are really exerting their effects through the voltage-gatedion channels of interest rather than through some unexpectednon-specific mechanism. One possibility for such control cells would beto use non-recombinant parent cells where the cells of the actualexperiment express the voltage-gated ion channels of interest due to therecombinant expression of those voltage-gated ion channels of interest.

Other types of controls would involve taking substances that areidentified by the methods of the present invention as activators orinhibitors of voltage-gated ion channels of interest and testing thosesubstances in the methods of the prior art in order to confirm thatthose substances are also activators and inhibitors when tested in thoseprior art methods.

One skilled in the art would recognize that, where the present inventioninvolves comparing control values for the flow of ions to test valuesfor the flow of ions and determining whether the control values aregreater or less than the test values, a non-trivial difference issought. For example, if in the methods of identifying inhibitors, thecontrol value were found to be 1% greater than the test value, thiswould not indicate that the substance is an inhibitor. Rather, oneskilled in the art would attribute such a small difference to normalexperimental variance. What is looked for is a significant differencebetween control and test values. For the purposes of this invention, asignificant difference fulfills the usual requirements for astatistically valid measurement of a biological signal. For example,depending upon the details of the experimental arrangement, asignificant difference might be a difference of at least 10%, preferablyat least 20%, more preferably at least 50%, and most preferably at least100%.

One skilled in the art would understand that the cells that give rise tothe control values need not be physically the same cells that give riseto the test values, although that is possible. What is necessary is thatthe cells that give rise to the control values be substantially the sametype of cell as the cells that give rise to the test values. A cell linethat has been transfected with and expresses a certain voltage-gated ionchannel could be used for both the control and test cells. Large numbersof such cells could be grown and a portion of those cells could beexposed to the substance and thus serve as the cells giving rise to thetest value for ion flow while a portion would not be exposed to thesubstance and would thus serve as the cells giving rise to the controlvalue for ion flow. No individual cell itself would be both control andtest cell but the virtual identity of all the cells in the cell lineensures that the methods would nevertheless be reliable.

“Substances” can be any substances that are generally screened in thepharmaceutical industry during the drug development process. Forexample, substances may be low molecular weight organic compounds (e.g.,having a molecular weight of less than about 1,000 daltons); RNA, DNA,antibodies, peptides, or proteins.

The conditions under which cells are exposed to substances in themethods described herein are conditions that are typically used in theart for the study of protein-ligand interactions: e.g., physiologicalpH; salt conditions such as those represented by such commonly usedbuffers as PBS or in tissue culture media; a temperature preferably ofabout 18° C. to about 45° C.; incubation times of from several secondsto several hours. Generally, the cells are present in wells in thesubstrate and the substances are added directly to the wells, optionallyafter first washing away the media in the wells.

Determining the values of ion flux in the methods of the presentinvention can be accomplished through the use of fluorescent indicatorcompounds. One type of fluorescent indicator compound is sensitive tothe level of intracellular calcium ions in the cells used in the presentinvention. This type of fluorescent indicator compound can be used whenthe methods are directed to those voltage-gated ion channels whoseactivity results in a change in intracellular calcium levels. Suchvoltage-gated ion channels include not only voltage-gated calciumchannels but also other types of voltage-gated ion channels where theactivity of those channels is naturally or can be coupled to changes inintracellular calcium levels. Many types of voltage-gated potassiumchannels can be so coupled. When using this approach to study avoltage-gated ion channel of interest that is not a voltage-gatedcalcium channel, it may be desirable to engineer the cells employed soas to recombinantly express voltage-gated calcium channels that arecoupled to the voltage-gated ion channel of interest.

Fluorescent indicator compounds suitable for measuring intracellularcalcium levels include various calcium indicator dyes (e.g., fura-2,fluo-3, fluo-4, indo-1, Calcium Green; see Veliçelebi et al., 1999,Meth. Enzymol. 294:20-47).

Calcium indicator dyes are substances which show a change in afluorescent characteristic upon binding calcium, e.g., greatly increasedintensity of fluorescence and/or a change in fluorescent spectra (i.e.,a change in emission or excitation maxima). Fluo-3, fura-2, and indo-1are commonly used calcium indicator dyes that were designed asstructural analogs of the highly selective calcium chelators ethyleneglycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) and1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Thefluorescence intensity from fluo-3 increases by more than 100-fold uponbinding of calcium. While the unbound dye exhibits very littlefluorescence, calcium-bound fluo-3 shows strong fluorescence emission at526 nm. Fura-2 is an example of a dye that exhibits a change in itsfluorescence spectrum upon calcium binding. In the unbound state, fura-2has an excitation maximum of 362 nm. This excitation maximum shifts to335 nm upon calcium binding, although there is no change in emissionmaximum. Binding of calcium to fura-2 can be monitored by excitation atthe two excitation maxima and determining the ratio of the amount offluorescence emission following excitation at 362 nm compared to theamount of fluorescence emission following excitation at 335 nm. Asmaller ratio (i.e., less emission following excitation at 362 nm)indicates that more fura-2 is bound to calcium, and thus a higherinternal calcium concentration in the cell.

The use of calcium indicator dyes entails loading cells with the dye, aprocess which can be accomplished by exposing cells to themembrane-permeable acetoxymethyl esters of the dyes. Once inside theplasma membrane of the cells, intracellular esterases cleave the esters,exposing negative charges in the free dyes. This prevents the free dyesfrom crossing the plasma membrane and thus leaves the free dyes trappedin the cells. Measurements of fluorescence from the dyes are then made,the cells are treated in such a way that the internal calciumconcentration is changed (e.g., by exposing cells to an activator orinhibitor of a voltage-gated ion channel), and fluorescence measurementsare again taken.

Fluorescence from the indicator dyes can be measured with a luminometeror a fluorescence imager. One preferred detection instrument is theFluorometric Imaging Plate Reader (FLIPR) (Molecular Devices, Sunnyvale,Calif.). The FLIPR is well suited to high throughput screening using themethods of the present invention as it incorporates integrated liquidhandling capable of simultaneously pipetting to 96 or 384 wells of amicrotiter plate and rapid kinetic detection using a argon laser coupledto a charge-coupled device imaging camera.

A typical protocol for use of calcium indicator dyes would entailputting cells expressing a voltage-gated ion channel of interest into anappropriate substrate (e.g., clear, flat-bottom, black-wall 96 wellplates) and allowing the cells to grow overnight in standard tissueculture conditions (e.g., 5% CO₂, 37° C.). The cells are generallyplated at a density of about 10,000 to 100,000 cells per well inappropriate growth medium. On the day of the assay, growth medium isremoved and dye loading medium is added to the wells.

If the calcium indicator dye is fluo-3, e.g., dye loading medium couldbe prepared by solubilizing 50 μg of fluo-3-AM ester (Molecular ProbesF-1242) in 22 μl DMSO to give a 2 mM dye stock. Immediately beforeloading the cells, 22 μl 20% pluronic acid (Molecular Probes P-3000) isadded to the dye. The tube containing the dye is mixed with a vortexmixer. For one 96-well plate, 44 ml of the dye/pluronic acid solution isadded to 10.5 ml of Hanks Balanced Salt Solution (Gibco/BRL Cat #14025-076) with 20 mM HEPES (Gibco/BRL Cat # 1560-080), and 1% fetalbovine serum (Gibco/BRL Cat # 26140-087; not BSA)). The dye and theloading medium are mixed by repeated inversion (final dye concentrationabout 4 μM).

Growth medium can be removed from the cells by washing (wash medium isHanks Balanced Salt Solution (Gibco/BRL Cat # 14025-076) with 20 mMHEPES (Gibco/BRL Cat # 1560-080), and 0.1% bovine serum albumin (SigmaCat # A-9647; not FBS) two times, leaving 100 μl residual medium in thewells after the second wash. Then 100 μl of the dye in the loadingmedium is added to each well. The cells are then incubated for 60minutes at 37° C. to allow for dye loading.

Following dye loading, the cells in each well are washed for four times,then fluorescent measurements of the cells are taken prior to exposureof the cells to substances that are to be tested. The cells are thenexposed to the substances and those substances that cause a change in afluorescent characteristic of the dye are identified. The measuringinstrument can be a fluorescent plate reader such as the FLIPR(Molecular Devices). Substances that cause a change in a fluorescentcharacteristic in the test cells but not the control cells are possibleactivators or inhibitors of the voltage-gated ion channel.

The exact details of the procedure outlined above are meant to beillustrative. One skilled in the art would be able to optimizeexperimental parameters (cell number, dye concentration, dye loadingtime, temperature of incubations, cell washing conditions, andinstrument settings, etc.) by routine experimentation depending on theparticular relevant experimental variables (e.g., type of cell used,identity of dye used). Several examples of experimental protocols thatcan be used are described in Veliçelebi et al., 1999, Meth. Enzymol.294:20-47. Other suitable instrumentation and methods for measuringtransmembrane potential changes via optical methods includesmicroscopes, multiwell plate readers and other instrumentation that iscapable of rapid, sensitive ratiometric fluorescence detection. Forexample, the VIPR (Aurora Biosciences, San Diego, Calif.) is anintegrated liquid handler and kinetic fluorescence reader for 96-welland greater multiwell plates. The VIPR reader integrates an eightchannel liquid handler, a multiwell positioning stage and a fiber-opticillumination and detection system. The system is designed to measurefluorescence from a column of eight wells simultaneously before, duringand after the introduction of liquid sample obtained from anothermicrotiter plate or trough. The VIPR reader excites and detects emissionsignals from the bottom of a multiwell plate by employing eighttrifurcated optical bundles (one bundle for each well). One leg of thetrifurcated fiber is used as an excitation source, the other two legs ofthe trifurcated fiber being used to detect fluorescence emission. A balllens on the end of the fiber increases the efficiency of lightexcitation and collection. The bifurcated emission fibers allow thereader to detect two emission signals simultaneously and are compatiblewith rapid signals generated by the FRET-based voltage dyes.Photomultiplier tubes then detect emission fluorescence, enablingsub-second emission ratio detection.

In particular embodiments, the calcium indicator dye is selected fromthe group consisting of: fluo-3, fura-2, fluo-4, fluo-5, calciumgreen-1, Oregon green, 488 BAPTA, SNARF-1, and indo-1.

In particular embodiments, the change in fluorescent characteristic isan increase in intensity of a fluorescence emission maximum. In otherembodiments, the change in fluorescent characteristic is a shift in thewavelength of an absorption maximum.

In particular embodiments, the cells naturally express the voltage-gatedion channel of interest. In other embodiments, the cells do notnaturally express the voltage-gated ion channel of interest but insteadhave been transfected with expression vectors that encode thevoltage-gated ion channel of interest so that the cells recombinantlyexpress the voltage-gated ion channel of interest. Transfection is meantto include any method known in the art for introducing expressionvectors into the cells. For example, transfection includes calciumphosphate or calcium chloride mediated transfection, lipofection,infection with a retroviral construct, and electroporation.

An alternative to the use of calcium indicator dyes is the use of theaequorin system. The aequorin system makes use of the proteinapoaequorin, which binds to the lipophilic chromophore coelenterazineforming a combination of apoaequorin and coelenterazine that is known asaequorin. Apoaequorin has three calcium binding sites and, upon calciumbinding, the apoaequorin portion of aequorin changes its conformation.This change in conformation causes coelenterazine to be oxidized intocoelenteramide, CO₂, and a photon of blue light (466 nm). This photoncan be detected with suitable instrumentation.

Since the gene encoding apoaequorin has been cloned (U.S. Pat. No.5,541,309; U.S. Pat. No. 5,422,266; U.S. Pat. No. 5,744,579; Inouye etal., 1985, Proc. Natl. Acad. Sci. USA 82:3154-3158; Prasher et al.,1985, Biochem. Biophys. Res. Comm. 126:1259-1268), apoaequorin can berecombinantly expressed in cells in which it is desired to measure theintracellular calcium concentration. Alternatively, existing cells thatstably express recombinant apoaequorin can be used. Such cells derivedfrom HEK293 cells and CHO-K1 cells are described in Button & Brownstein,1993, Cell Calcium 14:663-671. For example, the HEK293/aeq17 cell linecan be used as follows.

The HEK293/aeq17 cells are grown in Dulbecco's Modified Medium (DMEM,GIBCO-BRL, Gaithersburg, Md., USA) with 10% fetal bovine serum (heatinactivated), 1 mM sodium pyruvate, 500 μg/ml Geneticin, 100 μg/mlstreptomycin, 100 units/ml penicillin. Expression vectors encoding thevoltage-gated ion channel of interest as well as, optionally, thedesired voltage-gated calcium channel subunits (α_(1A), α_(1B), α_(1C),α_(1D), α_(1E), α_(1G), α_(1H), α_(1I), α₂δ, β₁, β₂, β₃, β₄, etc.) canbe transfected into the HEK293/aeq17 cells by standard methods in orderto express the desired voltage-gated ion channel subunits andvoltage-gated calcium channel subunits in the HEK293/aeq17 cells. Thecells are washed once with DMEM plus 0.1% fetal bovine serum, and thencharged for one hour at 37° C./5% CO₂ in DMEM containing 8 μMcoelenterazine cp (Molecular Probes, Eugene, Oreg., USA) and 30 μMglutathione. The cells are then washed once with Versene (GIBCO-BRL,Gaithersburg, Md., USA), detached using Enzyme-free cellissociationbuffer (GIBCO-BRL, Gaithersburg, Md., USA), diluted into ECB (Ham's F12nutrient mixture (GIBCO-BRL) with 0.3 mM CaCl₂, 25 mM HEPES, pH7.3, 0.1%fetal bovine serum). The cell suspension is centrifuged at 500×g for 5min. The supernatant is removed, and the pellet is resuspended in 10 mlECB. The cell density is determined by counting with a hemacytometer andadjusted to 500,000 cells/ml in ECB. The substances to be tested arediluted to the desired concentrations in ECB and aliquoted into theassay plates, preferably in triplicate, at 0.1 ml/well. The cellsuspension is injected at 0.1 ml/well, read and integrated for a totalof 400 readings using a luminometer (Luminoskan Ascent, Labsystems Oy,Helsinki, Finland). Alternatively, the cells may first be placed intothe assay plates and then the substances added. Data are analyzed usingthe software GraphPad Prism Version 3.0 (GraphPad Software, Inc., SanDiego, Calif., USA).

It will be understood by those skilled in the art that the procedureoutlined above is a general guide in which the various steps andvariables can be modified somewhat to take into account the specificdetails of the particular assay that is desired to be run. For example,one could use semisynthetic coelenterazine (Shimomura, 1989, Biochem. J.261:913-920; Shimomura et al., 1993, Cell Calcium 14:373-378); the timeof incubation of the cells with coelenterazine can be varied somewhat;somewhat greater or lesser numbers of cells per well can be used; and soforth.

For reviews on the use of aequorin, see Créton et al., 1999, MicroscopyResearch and Technique 46:390-397; Brini et al., 1995, J. Biol. Chem.270:9896-9903; Knight & Knight, 1995, Meth. Cell. Biol. 49:201-216. Alsoof interest may be U.S. Pat. No. 5,714,666 which describes methods ofmeasuring intracellular calcium in mammalian cells by the addition ofcoelenterazine co-factors to mammalian cells that express apoaequorin.

Another way to measure ion flow indirectly is to monitor changes intranscription that result from the activity of voltage-gated ionchannels by the use of transcription based assays. Transcription-basedassays involve the use of a reporter gene whose transcription is drivenby an inducible promoter whose activity is regulated by a particularintracellular event such as, e.g., changes in intracellular calciumlevels, that are caused by the activity of a voltage-gated ion channel.Transcription-based assays are reviewed in Rutter et al., 1998,Chemistry & Biology 5:R285-R290. Transcription-based assays of thepresent invention rely on the expression of reporter genes whosetranscription is activated or repressed as a result of intracellularevents that are caused by the interaction of a activator or inhibitorwith a voltage-gated ion channel.

An extremely sensitive transcription-based assay is disclosed inZlokarnik et al., 1998, Science 279:84-88 (Zlokarnik) and also in U.S.Pat. No. 5,741,657. The assay disclosed in Zlokarnik and U.S. Pat. No.5,741,657 employs a plasmid encoding β-lactamase under the control of aninducible promoter. This plasmid is transfected into cells together witha plasmid encoding a receptor for which it is desired to identifyagonists. The inducible promoter on the β-lactamase is chosen so that itresponds to at least one intracellular signal that is generated when anagonist binds to the receptor. Thus, following such binding of agonistto receptor, the level of β-lactamase in the transfected cellsincreases. This increase in β-lactamase is measured by treating thecells with a cell-permeable dye that is a substrate for cleavage byβ-lactamase. The dye contains two fluorescent moieties. In the intactdye, the two fluorescent moieties are physically linked, and thus closeenough to one another that fluorescence resonance energy transfer (FRET)can take place between them. Following cleavage of the dye into twoparts by β-lactamase, the two fluorescent moieties are located ondifferent parts, and thus can diffuse apart. This increases the distancebetween the fluorescent moieties, thus decreasing the amount of FRETthat can occur between them. It is this decrease in FRET that ismeasured in the assay.

The assay described in Zlokarnik and U.S. Pat. No. 5,741,657 can bemodified for use in the methods of the present invention by using aninducible promoter to drive β-lactamase where the promoter is activatedby an intracellular signal generated by the opening or closing of avoltage-gated ion channel. Cells expressing a voltage-gated ion channeland the inducible promoter-driven β-lactamase are placed in theapparatus of the present invention, where the open or closed state ofthe voltage-gated ion channels can be controlled. The cells are exposedto the cell-permeable dye and then exposed to substances suspected ofbeing activators or inhibitors of the voltage-gated ion channel. Thosesubstances that cause a change in the open or closed state of thevoltage-gated ion channel are identified by their effect on theinducible promoter-driven β-lactamase and thus on FRET. The induciblepromoter-driven β-lactamase is engineered with a suitable promoter sothat β-lactamase is induced when the substance is either an activator oran inhibitor, depending upon the nature of the assay.

The flow of ions through voltage-gated ion channels can also be measuredby measuring changes in membrane potential via the use of fluorescentvoltage sensitive dyes. The changes in membrane potential will depend onthe ion channels in the cell membrane. The resultant membrane potentialwill depend on the net properties of all the channels and the changecaused by inhibiting (through a substance that is an inhibitor orantagonist) or activating (through a substance that is an activator oran agonist) the voltage-gated ion channel of interest. One knowledgeablein cellular and membrane biophysics and electrophysiology willunderstand the directions of the changes in membrane potential sincethose changes depend on the ion channels present and the inhibition oractivation of those channels by test substances. In many cases whenusing fluorescent voltage sensitive dyes, the experimental system can becalibrated by using known activators or inhibitors of the voltage-gatedion channel of interest.

The present invention therefore includes assays that monitor changes inion flow caused by activators or inhibitors of voltage-gated ionchannels based upon FRET between a first and a second fluorescent dyewhere the first dye is bound to one side of the plasma membrane of acell expressing a voltage-gated ion channel of interest and the seconddye is free to move from one face of the membrane to the other face inresponse to changes in membrane potential. In certain embodiments, thefirst dye is impenetrable to the plasma membrane of the cells and isbound predominately to the extracellular surface of the plasma membrane.The second dye is trapped within the plasma membrane but is free todiffuse within the membrane. At normal (i.e., negative) restingpotentials of the membrane, the second dye is bound predominately to theinner surface of the extracellular face of the plasma membrane, thusplacing the second dye in close proximity to the first dye. This closeproximity allows for the generation of a large amount of FRET betweenthe two dyes. Following membrane depolarization, the second dye movesfrom the extracellular face of the membrane to the intracellular face,thus increasing the distance between the dyes. This increased distanceresults in a decrease in FRET, with a corresponding increase influorescent emission derived from the first dye and a correspondingdecrease in the fluorescent emission from the second dye. See FIG. 1 ofGonzález & Tsien, 1997, Chemistry & Biology 4:269-277. See also Gonzalez& Tsien, 1995, Biophys. J. 69:1272-1280 and U.S. Pat. No. 5,661,035.

In certain embodiments, the first dye is a fluorescent lectin or afluorescent phospholipid that acts as the fluorescent donor. Examples ofsuch a first dye are: a coumarin-labeled phosphatidylethanolamine (e.g.,N-(6-chloro-7-hydroxy-2-oxo-2H-1-benzopyran-3-carboxamidoacetyl)-dimyristoylphosphatidyl-ethanolamine)orN-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidylethanolamine);a fluorescently-labeled lectin (e.g., fluorescein-labeled wheat germagglutinin). In certain embodiments, the second dye is an oxonol thatacts as the fluorescent acceptor. Examples of such a second dye are:bis(1,3-dialkyl-2-thiobarbiturate)trimethineoxonols (e.g.,bis(1,3-dihexyl-2-thiobarbiturate)trimethineoxonol) orpentamethineoxonol analogues (e.g.,bis(1,3-dihexyl-2-thiobarbiturate)pentamethineoxonol; orbis(1,3-dibutyl-2-thiobarbiturate)pentamethineoxonol). See González &Tsien, 1997, Chemistry & Biology 4:269-277 for methods of synthesizingvarious dyes suitable for use in the present invention. In certainembodiments, the assay may comprise a natural carotenoid, e.g.,astaxanthin, in order to reduce photodynamic damage due to singletoxygen.

The use of such fluorescent dyes capable of moving from one face of theplasma membrane to the other is especially appropriate when the methodsof the present invention are directed to inwardly rectifying potassiumchannels. Activation of inwardly rectifying potassium channels resultsin increased potassium current flow across the plasma membrane. Thisincreased current flow results in a hyperpolarization of the cellmembrane that can be detected by use of the technique described abovesince such hyperpolarization will result in greater FRET.

In particular embodiments of the present invention, cells are utilizedthat have been transfected with expression vectors comprising DNA thatencodes a voltage-gated ion channel. Preferably, the cells do notnaturally express corresponding voltage-gated ion channels. For example,if the expression vectors direct the expression of a voltage-gatedcalcium channel, the cells will not naturally express voltage-gatedcalcium channels. Alternatively, if the cells naturally expresscorresponding voltage-gated ion channels, those correspondingvoltage-gated ion channels can be distinguished from the transfectedvoltage-gated ion channels in some manner, e.g., by the use ofappropriate inhibitors, by manipulation of membrane potential. Apreferred cell line for use in the present invention is the HEK293 cellline (ATCC 1573) since this cell line naturally expresses endogenouspotassium channels, which may be beneficial for electrical fieldstimulation experiments with channels that cause membrane potentialdepolarization (e.g., sodium or calcium channels).

In a specific embodiment, the subject invention relates to a C₁₋₆-37-3cell and cell line. The C₁₋₆-37-3 cell expresses the alpha1C calcium ionchannel subunit and the Kir 2.3 inward rectifying potassium channel onits plasma membrane.

Cells are generally eukaryotic cells, preferably mammalian cells. Thecells may be grown to the appropriate number on the substrates or theymay be placed on the substrate and used without further growth. Thecells may be attached to the substrate or, in those embodiments wherethe cells are placed or grown in wells, the cells may be suspensioncells that are suspended in the fluid in the wells. Primary cells orestablished cell lines may be used.

Suitable cells for transfection with expression vectors that direct theexpression of voltage-gated ion channels include but are not limited tocell lines of human, bovine, porcine, monkey and rodent origin. Thecells may be adherent or non-adherent. Cells and cell lines which aresuitable and which are widely available, include but are not limited to:L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), HEK293(ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92),NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616),BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), CPAE (ATCC CCL 209), Saos-2(ATCC HTB-85), ARPE-19 human retinal pigment epithelium (ATCC CRL-2302),GH3 cells, T-REx-293 cells (Invitrogen, R710-07), T-REx-CHO cells(Invitrogen, R718-07) and primary cardiac myocytes.

A variety of voltage-gated ion channels may be used in the presentinvention. For example, voltage-gated sodium channels, voltage-gatedpotassium channels, and voltage-gated calcium channels are suitable.

In certain embodiments of the present invention, the cells used do notnaturally express the voltage-gated ion channel of interest. Instead,DNA encoding the voltage-gated ion channel is transfected into cells inorder to express the voltage-gated ion channel in the plasma membrane ofthe cells. DNA encoding voltage-gated ion channels can be obtained bymethods well known in the art. For example, a cDNA fragment encoding avoltage-gated ion channel can be isolated from a suitable cDNA libraryby using the polymerase chain reaction (PCR) employing suitable primerpairs. The cDNA fragment encoding the voltage-gated ion channel can thenbe cloned into a suitable expression vector. Primer pairs can beselected based upon the known DNA sequence of the voltage-gated ionchannel it is desired to obtain. Suitable cDNA libraries can be madefrom cellular or tissue sources known to contain mRNA encoding thevoltage-gated ion channel.

One skilled in the art would know that for certain voltage-gated ionchannels, it is desirable to transfect, and thereby express, more thanone subunit in order to obtain a functional voltage-gated ion channel.For example, N-type calcium channels are composed of a multisubunitcomplex containing at least an α1B, an α2δ, and a β1 subunit. On theother hand, T-type calcium channels are functional with only a singlesubunit, e.g., α1G, α1H, or all. Common knowledge in the art of thesubunit composition of a voltage-gated ion channel of interest will leadthe skilled artisan to express the correct subunits in the transfectedcells. U.S. Pat. No. 5,851,824 provides sequences for thealpha.-1C/alpha-1D, alpha-2, β-1, and gamma.subunits

One skilled in the art could use published voltage-gated ion channelsequences to design PCR primers and published studies of voltage-gatedion channel expression to select the appropriate sources from which tomake cDNA libraries in order to obtain DNA encoding the voltage-gatedion channels. The following publications may be of use in this regard:

-   -   U.S. Pat. No. 5,876,958;    -   U.S. Pat. No. 6,096,514;    -   U.S. Pat. No. 6,090,623

Hondeghem, L. M., Katzung, B. G. (1984) Antiarrhythmic agents: themodulated receptor mechanism of action of sodium and calciumchannel-blocking drugs. Annu-Rev-Pharmacol-Toxicol. 24:387-423.; Zheng,W., Stoltefuss, J., Goldmann, S., and Triggle, D. J. (1992)Pharmacologic and radioligand binding studies of 1,4-dihydropyridines inrat cardiac and vasculr preparations: stereoselectivity and voltagedependence of antagonist and activator interactions. Mol. Pharmacol.41(3):535-541.; and Triggle, D. J., Hawthorn, M. H. and Zheng, W. (1988)Potential-dependent interactions of nitrendipine and related1,4-dihydropyridines in functional smooth muscle preparations. J.Cardiovasc. Pharmacol., 12 (Suppl.4):s91-s93.

The following table provides a list of known ion channels andinformation concerning each: TABLE 1 Some ion channel genes of interestfor ion flux experiments Cytogenetic MIM PubMed Symbol Full NameLocation Number ID SCN1 symbol withdrawn, see SCN1A SCN1A sodiumchannel, voltage-gated, type I, 2q24 182389 8062593 alpha polypeptideSCN1B sodium channel, voltage-gated, type I, beta 19 600235 8394762polypeptide SCN2A1 sodium channel, voltage-gated, type II, 2q22-q23182390 1317301 alpha 1 polypeptide SCN2A2 sodium channel, voltage-gated,type II, 2q23-q24 601219 1317301 alpha 2 polypeptide SCN2A symbolwithdrawn, see SCN2A1 — SCN2B sodium channel, voltage-gated, type II,11q22-qter 601327 10198179 beta polypeptide SCN3A sodium channel,voltage-gated, type III, 2q24 182391 9589372 alpha polypeptide SCN4Asodium channel, voltage-gated, type IV, 17q23-q25.3 603967 1654742 alphapolypeptide SCN4B sodium channel, voltage-gated, type IV, reserved betapolypeptide SCN5A sodium channel, voltage-gated, type V, 3p21 600163alpha polypeptide (long (electrocardiographic) QT syndrome 3) SCN6Asodium channel, voltage-gated, type VI, 2q21-q23 182392 10198179 alphapolypeptide SCN7A symbol withdrawn, see SCN6A — SCN8A sodium channel,voltage gated, type VIII, 12q13.1 600702 7670495 alpha polypeptide SCN9Asodium channel, voltage-gated, type IX, 2q24 603415 7720699 alphapolypeptide SCN10A sodium channel, voltage-gated, type X, 3p21-p22604427 9839820 alpha polypeptide SCN11A sodium channel, voltage-gated,type XI, 3p21-p24 604385 10444332 alpha polypeptide SCN12A sodiumchannel, voltage-gated, type XII, 3p23-p21.3 10623608 alpha polypeptideSCNN1 symbol withdrawn, see SCNN1A — SCNN1A sodium channel,nonvoltage-gated 1 alpha 12p13 600228 7896277 SCNN1B sodium channel,nonvoltage-gated 1, beta 16p12.2-p12.1 600760 (Liddle syndrome) SCNN1Dsodium channel, nonvoltage-gated 1, delta 1p36.3-p36.2 601328 8661065SCNN1G sodium channel, nonvoltage-gated 1, 16p12 600761 7490094 gammaCACNA1A calcium channel, voltage-dependent, P/Q 19p13 601011 8825650type, alpha 1A subunit CACNA1B calcium channel, voltage-dependent, L9q34 601012 8825650 type, alpha 1B subunit CACNA1C calcium channel,voltage-dependent, L 12pter-p13.2 114205 1650913 type, alpha 1C subunitCACNA1D calcium channel, voltage-dependent, L 3p14.3 114206 1664412type, alpha 1D subunit CACNA1E calcium channel, voltage-dependent, alpha1q25-q31 601013 8388125 1E subunit CACNA1F calcium channel,voltage-dependent, alpha Xp11.23-p11.22 300110 9344658 1F subunitCACNA1G calcium channel, voltage-dependent, alpha 17q22 604065 94953421G subunit CACNA1H calcium channel, voltage-dependent, alpha 16p13.39670923 1H subunit CACNA1I calcium channel, voltage-dependent, alpha22q12.3-13.2 10454147 1I subunit CACNA1S calcium channel,voltage-dependent, L 1q31-q32 114208 7916735 type, alpha 1S subunitCACNA2 symbol withdrawn, see CACNA2D1 — CACNA2D1 calcium channel,voltage-dependent, alpha 7q21-q22 114204 8188232 2/delta subunit 1CACNA2D2 calcium channel, voltage-dependent, alpha reserved 2/deltasubunit 2 CACNB1 calcium channel, voltage-dependent, beta 1 17q21-q22114207 8381767 subunit CACNB2 calcium channel, voltage-dependent, beta 210p12 600003 9254841 subunit CACNB3 calcium channel, voltage-dependent,beta 3 12q13 601958 8119293 subunit CACNB4 calcium channel,voltage-dependent, beta 4 2q22-q31 601949 9628818 subunit CACNG1 calciumchannel, voltage-dependent, 17q24 114209 8395940 gamma subunit 1 CACNG2calcium channel, voltage-dependent, reserved 602911 gamma subunit 2CACNG3 calcium channel, voltage-dependent, reserved gamma subunit 3CACNG4 calcium channel, voltage-dependent, 17q24 10613843 gamma subunit4 CACNG5 calcium channel, voltage-dependent, 17q24 10613843 gammasubunit 5 CACNG6 calcium channel, voltage-dependent, 19q13.4 11170751gamma subunit 6 CACNG7 calcium channel, voltage-dependent, 19q13.411170751 gamma subunit 7 CACNG8 calcium channel, voltage-dependent,19q13.4 11170751 gamma subunit 8 KCNA1 potassium voltage-gated channel,shaker- 12p13 176260 1349297 related subfamily, member 1 (episodicataxia with myokymia) KCNA1B literature alias, see KCNAB1 — KCNA2potassium voltage-gated channel, shaker- 12 176262 related subfamily,member 2 KCNA2B literature alias, see KCNAB2 — KCNA3 potassiumvoltage-gated channel, shaker- 1p13.3 or 13 176263 2251283 relatedsubfamily, member 3 KCNA3B literature alias, see KCNAB3 — KCNA4potassium voltage-gated channel, shaker- 11p14 176266 2263489 relatedsubfamily, member 4 KCNA4L potassium voltage-gated channel, shaker-11q14 8449523 related subfamily, member 4-like KCNA5 potassiumvoltage-gated channel, shaker- 12 176267 related subfamily, member 5KCNA6 potassium voltage-gated channel, shaker- reserved 176257 relatedsubfamily, member 6 KCNA7 potassium voltage-gated channel, shaker- 19176268 related subfamily, member 7 KCNA8 literature alias, see KCNQ1 —KCNA9 symbol withdrawn, see KCNQ1 — KCNA10 potassium voltage-gatedchannel, shaker- reserved 602420 related subfamily, member 10 KCNAB1potassium voltage-gated channel, shaker- 3q26.1 601141 8838324 relatedsubfamily, beta member 1 KCNAB2 potassium voltage-gated channel, shaker-1p36.3 601142 8838324 related subfamily, beta member 2 KCNAB3 potassiumvoltage-gated channel, shaker- 17p13.1 604111 9857044 related subfamily,beta member 3 KCNB1 potassium voltage-gated channel, Shab- 20q13.2600397 7774931 related subfamily, member 1 KCNB2 potassium voltage-gatedchannel, Shab-  8 9612272 related subfamily, member 2 KCNC1 potassiumvoltage-gated channel, Shaw- 11p15 176258 8449507 related subfamily,member 1 KCNC2 potassium voltage-gated channel, Shaw- 12 and 1762568111118 related subfamily, member 2 19q13.4 KCNC3 potassiumvoltage-gated channel, Shaw- 19 176264 1740329 related subfamily, member3 KCNC4 potassium voltage-gated channel, Shaw- 1p21 176265 1920536related subfamily, member 4 KCND1 potassium voltage-gated channel, Shal-Xp11.23-p11.3 300281 10729221 related subfamily, member 1 KCND2potassium voltage-gated channel, Shal- 7q31-32 605410 10551270 relatedsubfamily, member 2 KCND3 potassium voltage-gated channel, Shal- 1p13.2605411 10942109 related subfamily, member 3 KCNE1 potassiumvoltage-gated channel, Isk- 21q22.1-q22.2 176261 8432548 related family,member 1 KCNE1L potassium voltage-gated channel, Isk- Xq22.3 30032810493825 related family, member 1-like KCNE2 potassium voltage-gatedchannel, Isk- 21q22.1 603796 10219239 related family, member 2 KCNE3potassium voltage-gated channel, Isk- reserved 604433 10219239 relatedfamily, member 3 KCNE4 potassium voltage-gated channel, Isk- reserved10219239 related family, member 4 KCNF1 potassium voltage-gated channel,2p25 603787 9434767 subfamily F, member 1 KCNF2 literature alias, seeKCNG2 — KCNF symbol withdrawn, see KCNF1 — KCNG1 potassium voltage-gatedchannel, 20q13 603788 9434767 subfamily G, member 1 KCNG2 potassiumvoltage-gated channel, 18q22-18q23 605696 10551266 subfamily G, member 2KCNG symbol withdrawn, see KCNG1 — KCNH1 potassium voltage-gatedchannel, 1q32-41 603305 9738473 subfamily H (eag-related), member 1KCNH2 potassium voltage-gated channel, 7q35-q36 152427 7842012 subfamilyH (eag-related), member 2 KCNH3 potassium voltage-gated channel, 12q13604527 10455180 subfamily H (eag-related), member 3 KCNH4 potassiumvoltage-gated channel, reserved 604528 10455180 subfamily H(eag-related), member 4 KCNH5 potassium voltage-gated channel, 14 6057169738473 subfamily H (eag-related), member 5 KCNIP1 Kv channelinteracting protein 1 reserved 10676964 KCNIP2 Kv channel-interactingprotein 2 10 10676964 KCNIP3 literature alias, see CSEN — KCNJ1potassium inwardly-rectifying channel, 11q24 600359 7680431 subfamily J,member 1 KCNJ2 potassium inwardly-rectifying channel, 17q23.1-q24.2600681 7696590 subfamily J, member 2 KCNJ3 potassium inwardly-rectifyingchannel, 2q24.1 601534 8088798 subfamily J, member 3 KCNJ4 potassiuminwardly-rectifying channel, 22q13.1 600504 8016146 subfamily J, member4 KCNJ5 potassium inwardly-rectifying channel, 11q24 600734 subfamily J,member 5 KCNJ6 potassium inwardly-rectifying channel, 21q22.1 6008777796919 subfamily J, member 6 KCNJ7 symbol withdrawn, see KCNJ6 — KCNJ8potassium inwardly-rectifying channel, 12p11.23 600935 8595887 subfamilyJ, member 8 KCNJ9 potassium inwardly-rectifying channel, 1q21-1q23600932 8575783 subfamily J, member 9 KCNJ10 potassiuminwardly-rectifying channel, 1q 602208 9367690 subfamily J, member 10KCNJ11 potassium inwardly-rectifying channel, 11p15.1 600937 7502040subfamily J, member 11 KCNJ12 potassium inwardly-rectifying channel,17p11.1 602323 7859381 subfamily J, member 12 KCNJ13 potassiuminwardly-rectifying channel, 2q37 603208 9878260 subfamily J, member 13KCNJ14 potassium inwardly-rectifying channel, 19q13 603953 9592090subfamily J, member 14 KCNJ15 potassium inwardly-rectifying channel,21q22.2 602106 9299242 subfamily J, member 15 KCNJ16 potassiuminwardly-rectifying channel, 17q23.1-q24.2 605722 11240146 subfamily J,member 16 KCNJN1 channel, subfamily J, inhibitor 1 17p11.2-p11.1 6026048647284 KCNK1 potassium channel, subfamily K, member 1q42-q43 6017458661042 1 (TWIK-1) KCNK2 potassium channel, subfamily K, member 1q41603219 9721223 2 (TREK-1) KCNK3 potassium channel, subfamily K, member2p23 603220 9312005 3 (TASK-1) KCNK4 potassium inwardly-rectifyingchannel, 11q13 605720 10767409 subfamily K, member 4 KCNK5 potassiumchannel, subfamily K, member 6p21 603493 9812978 5 (TASK-2) KCNK6potassium channel, subfamily K, member 19q13.1 603939 10075682 6(TWIK-2) KCNK7 potassium channel, subfamily K, member 7 11q13 60394010206991 KCNK9 potassium channel, subfamily K, member  8 605874 107340769 (TASK-3) KCNK10 potassium channel, subfamily K, member reserved 60587310 KCNK12 potassium channel, subfamily K, member 2p22-2p21 12 KCNK13potassium channel, subfamily K, member 14q24.1-14q24.3 11060316 13KCNK14 potassium channel, subfamily K, member 2p22-2p21 11060316 14KCNK15 potassium channel, subfamily K, member reserved 15 KCNMA1potassium large conductance calcium- 10 600150 7987297 activatedchannel, subfamily M, alpha member 1 KCNMB1 potassium large conductancecalcium- 5q34 603951 8799178 activated channel, subfamily M, beta member1 KCNMB2 symbol withdrawn, see KCNMB3 — KCNMB2 potassium largeconductance calcium- reserved 605214 10097176 activated channel,subfamily M, beta member 2 KCNMB2L symbol withdrawn, see KCNMB3L —KCNMB3 potassium large conductance calcium- 3q26.3-q27 605222 10585773activated channel, subfamily M beta member 3 KCNMB3L potassium largeconductance calcium- 22q11 10585773 activated channel, subfamily M, betamember 3-like KCNMB4 potassium large conductance calcium- reserved605223 activated channel, subfamily M, beta member 4 KCNMBL symbolwithdrawn, see KCNMB3 — KCNMBLP symbol withdrawn, see KCNMB3L — KCNN1potassium intermediate/small conductance 19p13.1 602982 8781233calcium-activated channel, subfamily N, member 1 KCNN2 potassiumintermediate/small conductance reserved 605879 calcium-activatedchannel, subfamily N, member 2 KCNN3 potassium intermediate/smallconductance 22q11-q13.1 602983 9491810 calcium-activated channel,subfamily N, member 3 KCNN4 potassium intermediate/small conductance19q13.2 602754 9380751 calcium-activated channel, subfamily N, member 4KCNQ1 potassium voltage-gated channel, KQT- 11p15.5 192500 8528244 likesubfamily, member 1 KCNQ1OT1 KCNQ1 overlapping transcript 1 11p15.5604115 10220444 KCNQ2 potassium voltage-gated channel, KQT- 20q13.3-2121200 9425895 like subfamily, member 2 20q13.3 KCNQ3 potassiumvoltage-gated channel, KQT- 8q24 121201 9425900 like subfamily, member 3KCNQ4 potassium voltage-gated channel, KQT- 1p34 603537 10025409 likesubfamily, member 4 KCNQ5 potassium voltage-gated channel, KQT- 6q1410787416 like subfamily, member 5 KCNS1 potassium voltage-gated channel,delayed- reserved 602905 9305895 rectifier, subfamily S, member 1 KCNS2potassium voltage-gated channel, delayed- 8q22 602906 9305895 rectifier,subfamily S, member 2 KCNS3 potassium voltage-gated channel, delayed-reserved 603888 10484328 rectifier, subfamily S, member 3

PCR reactions can be carried out with a variety of thermostable enzymesincluding but not limited to AmpliTaq, AmpliTaq Gold, or Ventpolymerase. For AmpliTaq, reactions can be carried out in 10 mM Tris-Cl,pH 8.3, 2.0 mM MgCl₂, 200 μM of each dNTP, 50 mM KCl, 0.2 M of eachprimer, 10 ng of DNA template, 0.05 units/μl of AmpliTaq. The reactionsare heated at 95° C. for 3 minutes and then cycled 35 times usingsuitable cycling parameters, including, but not limited to, 95° C., 20seconds, 62° C., 20 seconds, 72° C., 3 minutes. In addition to theseconditions, a variety of suitable PCR protocols can be found in PCRPrimer. A Laboratory Manual, edited by C. W. Dieffenbach and G. S.Dveksler, 1995, Cold Spring Harbor Laboratory Press; or PCR Protocols: AGuide to Methods and Applications, Michael et al., eds., 1990, AcademicPress.

It is desirable to sequence the DNA encoding voltage-gated ion channelsobtained by the herein-described methods, in order to verify that thedesired voltage-gated ion channel has in fact been obtained and that nounexpected changes have been introduced into its sequence by the PCRreactions. The DNA can be cloned into suitable cloning vectors orexpression vectors, e.g., the mammalian expression vector pcDNA3.1(Invitrogen, San Diego, Calif.) or other expression vectors known in theart or described herein.

A variety of expression vectors can be used to recombinantly express DNAencoding voltage-gated ion channels for use in the present invention.Commercially available expression vectors which are suitable include,but are not limited to, pMC1neo (Stratagene), pSG5 (Stratagene), pcDNAIand pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen, San Diego, Calif.),EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198),pCI.neo (Promega), pTRE (Clontech, Palo Alto, Calif.), pV1Jneo, pIRESneo(Clontech, Palo Alto, Calif.), pCEP4 (Invitrogen, San Diego, Calif.),pSC11, and pSV2-dhfr (ATCC 37146). The choice of vector will depend uponcell type in which it is desired to express the voltage-gated ionchannels, as well as on the level of expression desired, and the like.

The expression vectors can be used to transiently express or stablyexpress the voltage-gated ion channels. The transient expression orstable expression of transfected DNA is well known in the art. See,e.g., Ausubel et al., 1995, “Introduction of DNA into mammalian cells,”in Current Protocols in Molecular Biology, sections 9.5.1-9.5.6 (JohnWiley & Sons, Inc.).

As an alternative to the above-described PCR methods, cDNA clonesencoding ion channels can be isolated from cDNA libraries using as aprobe oligonucleotides specific for the desired voltage-gated ionchannels and methods well known in the art for screening cDNA librarieswith oligonucleotide probes. Such methods are described in, e.g.,Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.),1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K.,Vol. I, II. Oligonucleotides that are specific for particularvoltage-gated ion channels and that can be used to screen cDNA librariescan be readily designed based upon the known DNA sequences of thevoltage-gated ion channels and can be synthesized by methods well-knownin the art.

EXAMPLE 1

Immunofluorescence staining was all performed at room temperature. Cellswere washed three times with Dulbecco's phosphate buffered saline(D-PBS) and then fixed with 4% paraformldehyde for 30 min. After threewashes with D-PBS, the cells were blocked and permeabilized with TBS (10mM Tris-HCl, pH 7.5, and 150 mM NaCl) containing 4% nonfat dry milk and0.1% Triton X-100 for 1 hr, and incubated with the affinity purifiedpolyclonal antibodies against human alpha 1C or kir2.3 for 1 hr. Thenthe cells were washed three times with TBS and incubated with thesecondary antibody (Cy3-conjugated anti-rabbit IgG, at 1:250, JacksonImmunoResearch, PA) for 1 hr. The cells were finally washed with D-PBSthree times and viewed under indirect immunofluorescence on a ZeissAxioskop microscope. FIGS. 1 and 2 show that cells were successfullytransfected and expressing calcium and potassium channels, respectively,on their plasma membranes.

EXAMPLE 2

Hek 293 cells were stably transfected with the alpha 1C subunit of theL-type Calcium ion channel and Kir 2.3 inward K⁺ rectifying channel(C1-6-37-3 cells). Calcium influx into the cells was measured in aFLIPR™ (Molecular Devices, CA). The C1-6-37-3 cells were seeded intoblack 96 well plates with clear bottoms coated with poly-D-lysine atdensity of 50000 cells/well and cultured overnight. Next day the cellswere washed twice with assay buffer containing 137 mM NaCl; 0.34 mMNa₂HPO₄; 4.2 mM NaHCO₃; 0.44 mM KH₂PO₄; 0.41 mM MgSO₄; 0.49 mM MgCl²; 20mM HEPES; 5.5 mM D-glucose and 0.1% BSA and incubated with Fluo-3AM(final concentration 4 μM, Molecular probe) for 1 hr at 37° C., 5% CO₂and 95% O₂. After cells were washed four times either with restingcondition (5.8 K⁺) or depolarized condition (30 mM K⁺), the cell platewas placed into the FLIPR™ to monitor cell fluorescence (λ_(EX)=488 nm,λ_(EM)=540 nm) before and after the addition of calcium blockers andagonists (final 85.8 mM K⁺).

Cellular membrane potentials were measured using an Axopatch 200B patchamplifier (Axon Instruments Inc., Foster City, Calif.) in current clampmore using the “perforated patch” clamp method (Horn and Korn). Thepatch pipette contained (in mM): 120 KMeS04, 20 KCl, 9 Mg2Cl, 10 HEPES,Nystatin 200 μg/ml, pH 7.3. The bath solution contained (in mM): 140NaCl, 1.2 Mg2Cl, 10 HEPES, 1.3 Ca2Cl, 21 D-glucose, pH 7.4. Standardelectrophysiological methods were employed. Changes in extracellularpotassium were made by additions of a concentrated stock to the standardbath solution to the appropriate dilution.

Results:

Table 2 shows the membrane potential of the C1-6-37-3 cells recorded atvarious extracellular potassium concentrations. This experiment confirmsthat changes in potassium alter the membrane potential of these cellsapproximately as predicted by the Nernst equation.

FIG. 4 shows that calcium influx into fluo-3 loaded cells in response toincreasing potassium concentration was concentration dependent andpossessed an EC₅₀ of 11 mM K⁺. The potency of the inhibitory effect ofnimodipine and other calcium channel antagonists on calcium influxthrough the 1αC channel was shown to depend on membrane potential (table3, FIGS. 5-7). Preincubation of cells with 30 mM K⁺ (Vm=−28 mV)increased the potency of nimodipine to block calcium influx compared tothe preincubation of these cells with 5.8 mM K⁺ (Vm=−65 mV). This assaycaptures the state-dependent interactions of 1,4-dihydropyridines thathave been identified previously. TABLE 2 Membrane potential of C1-6-37-3cell line recorded in various potassium concentrations using Nystatinperforated patch [K]_(out) mM Resting potential n 0.4 −99.3 ± 10.6   64.0 −73 ± 0.7  6 5.8 −64.7 ± 2.6    7 30 −27.6 ± 2.4    7 80 7.5 ± 7.1 7Values are the mean ±

TABLE 3 IC50 (nM) values of calcium channel antagonists for inhibitionof K⁺-induced calcium influx either in 30 mM K⁺ (depolarized condition)or 5.8 mM K⁺ (resting condition). Antagonists 5.8 mM [K]o n 30 mM [K]o nF Nimodipine 59 ± 27 4 3 ± 3 5 21 Nifedipine 43 ± 12 4 7 ± 1 3 7Nitrendipine 51 ± 18 4 6 ± 3 2 8 Mibefradil 3458 ± 867  4 791 ± 43  5 4Values are the mean ± S.D.F indicates the ratio of the IC50 values of 5.8 mM K⁺ and 30 mM K⁺.

EXAMPLE 3

Cellular membrane potentials were measured using an Axopatch 200B patchamplifier (Axon Instruments Inc., Foster City, Calif.) in current clampmore using the ‘perforated patch’ clamp method (Horn and Korn). Thepatch pipette contained (in mM): 120 KMeSO₄, 20 KCl, 9 Mg2Cl, 10 HEPES,Nystatin 200 μg/ml, pH7.3. The bath solution contained (in mM): 140NaCl, 1.2 Mg2Cl, 10 HEPES, 1.3 Ca2Cl, 21 D-glucose, pH7.4. Standardelectrophysiological methods were employed. Changes in extracellularpotassium were made by additions of a concentrated stock to the standardbath solution to the appropriate dilution. FIG. 3 shows the relationshipbetween extracellular potassium ([K]o) and cell membrane potential.Three situations are shown. One is the prediction of the Nernst equationfor a perfectly K-selective membrane. The other curves show the effectsof partial permeability by other ions, Na⁺ and/or Cl⁻. Membranepotential can be set in a non-voltage clamped cell by adjusting externalpotassium. A cell line expressing an inward rectifier K channel (Kir2.3)to set the resting membrane potential will permit control of membraneresting potential by extracellular potassium.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties to the extent notinconsistent with the teachings herein. All patents, patentapplications, publications, texts and references discussed or citedherein are understood to be incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually set forth in its entirety. In addition, all references,patents, applications, and other documents cited in an InventionDisclosure Statement, Examiner's Summary of Cited References, orotherwise entered into the file history of this application are taken tobe incorporated by reference into this specification for the benefit oflater applications claiming priority to this application. Finally, allterms not specifically defined are first taken to have the meaning giventhrough usage in this disclosure, and if no such meaning is inferable,their normal meaning.

1. A method for testing a compound for activity as an agonist orantagonist of a calcium channel, comprising the steps of: (a) contactinga cell expressing a functional voltage-gated calcium ion channel and afunctional potassium ion channel with a solution having a potassiumconcentration where the membrane potential of the cell is modulatedwithout fully depolarizing the cell; (b) simultaneous to or subsequentto step (a), contacting the cell with (i) a substance of interest and(ii) an ion or molecule capable of entering the cell through afunctional calcium channel; (c) depolarizing the cell membrane of thecell; (d) detecting the channel mediated ion flux into the cell; and (e)comparing the ion flux thus detected from step (d) to an ion fluxproduced in a control experiment, wherein the control experimentcomprises subjecting a separate cell to the steps (a), (b)(ii), (c) and(d), but not step (b)(i); where a difference in ion flux detected instep (d) and the control experiment indicates that the substance ofinterest is an agonist or antagonist of a calcium channel.
 2. A methodof identifying state-dependent antagonists of a voltage-gated calciumion channel comprising: (a) providing a divided tissue culture platecomprising individual compartments, where at least two of the individualcompartments contain living eukaryotic cells that express a plurality offunctional voltage-gated calcium ion channels and functional potassiumchannels on their plasma membranes, the cytoplasm of the cellscomprising an ion-sensitive fluorescent indicator compound; (b)adjusting the membrane potential of the cells by altering extracellularpotassium concentration in at least one of the compartments containingthe cells; (c) adding a substance of interest to at least one of theindividual compartments containing the cells; (d) depolarizing the cellsin the at least two compartments containing cells, wherein at least onecompartment is subjected to step (c), test group, and at least onecompartment is not subjected to step (c), control group; (e) detectingthe ion flux into the cells of step (d); and (f) comparing the ion fluxinto the cells of the test group with the cells of the control group;where if the value of ion flux in the test group cells is lower than thecontrol group cells, the substance is an antagonist of the voltage-gatedcalcium ion channel.
 3. The method of claim 2, wherein the dividedtissue culture plate is a multiwell tissue culture plate comprising atleast two wells.
 4. The method of claim 3, wherein the multiwell tissueculture plate comprises 12, 24, 96, 384, 1,536, or 3,456 wells.
 5. Themethod of claim 2, wherein at least 10 substances are tested in a 24hour period.
 6. The method of claim 2 where the cells are selected fromthe group consisting of: L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M(ATCC CCL 1.2), HEK293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCCCCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2),C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), CPAE(ATCC CCL 209), Saos-2 (ATCC HTB-85), ARPE-19 human retinal pigmentepithelium (ATCC CRL-2302), GH3 cells, TREx-292 cells, T-REx-CHO cells,and primary cardiac myocytes.
 7. The method of claim 2, where the cellsare HEK293 cells stably transfected to express the alpha-1C subunit ofthe voltage-gated calcium ion channel and Kir 2.3 inward-rectifyingpotassium channel.
 8. The method of claim 2 wherein the fluorescentindicator compound is selected from the group consisting of fluo-3,fura-2, fluo-4, fluo-5, calcium green-1, Oregon green, 488 BAPTA,SNARF-1, and indo-1.
 9. The method of claim 2, wherein the substance isidentified as an antagonist when the current flow into the cells of thetest group is lower than the current flow into the cells of the controlgroup.
 10. The method of claim 2, wherein the detecting step (e) employsa fluorescence or luminescence indicator device.
 11. The method of claim2, wherein the detecting step (e) employs a FLIPR or VIPR device.
 12. Amethod of identifying state-dependent antagonists of a voltage-gatedcalcium ion channel comprising: (a) providing a divided tissue cultureplate comprising individual compartments, where at least two of theindividual compartments contain living eukaryotic cells that express aplurality of functional alpha 1C calcium ion channels and functional Kir2.3 inward rectifying potassium channels on their plasma membranes, thecytoplasm of the cells comprising an ion-sensitive fluorescent indicatorcompound; (b) adjusting the membrane potential of the cells by alteringextracellular potassium concentration in at least one of thecompartments containing the cells; (c) adding a substance of interest toat least one of the individual compartments containing the cells; (d)depolarizing the cells in the at least two compartments containingcells, wherein at least one compartment is subjected to step (c), testgroup, and at least one compartment is not subjected to step (c),control group; (e) detecting the ion flux into the cells of step (d);and (f) comparing the ion flux into the cells of the test group with thecells of the control group; where if the value of ion flux in the testgroup cells is lower than the control group cells, the substance is anantagonist of the voltage-gated calcium ion channel.
 13. The method ofclaim 12 further comprising comparing ion flux in the test group withthat in a second test group, the second test group comprising cellssubjected to steps (b) and (c), but whose membrane potentials have beenadjusted to a value different than that of the test group cells; whereif the value of ion flux in the test group cells is different than thevalue of ion flux in the second test group cells, then the substancepossesses a state-dependent potency on the voltage-gated calcium ionchannel.
 14. A method of identifying antagonists possessingstate-dependent potency for a voltage-gated calcium ion channelcomprising: (a) providing a divided tissue culture plate comprisingindividual compartments, where at least two of said individualcompartments contain living eukaryotic cells that express a plurality offunctional voltage-gated calcium ion channels and functional potassiumchannels on their plasma membranes, the cytoplasm of said cellscomprising an ion-sensitive fluorescent indicator compound; (b)adjusting the membrane potential of a first group of cells and a secondgroup of cells by altering extracellular potassium concentration in theindividual compartments containing said first and second group of cell,wherein the membrane potential of the second test group cells is lowerthan the value of the first test group cells; (c) adding a substance ofinterest to the individual compartments containing the first and secondgroups of cells; (d) depolarizing the cells in the at least twocompartments containing cells; (e) detecting the ion flux into the cellsof step (d); and (f) comparing the ion flux in the cells of the firsttest group with the cells of the second test group; where if the valueof ion flux in the first test group cells is different than the value ofion flux in the second test group cells, then the substance possessesstate-dependent potency for said voltage-gated calcium ion channel.